Several forms of arrayed hybridization reactions are currently being developed under the common rubric of “sequencing by hybridization” (SBH). Included are “format 1” versions of SBH, involving stepwise hybridization of different oligonucleotide probes with arrays of DNA samples gridded onto membranes, and “format 2” implementations, involving hybridization of a single nucleic acid “target sequence” to an array of oligonucleotide probes tethered to a flat surface or immobilized within a thin gel matrix. The term “genosensor” has heretofore referred to a form of SBH in which oligonucleotides are tethered to a surface in a two-dimensional array and serve as recognition elements for complementary sequences present in a nucleic acid “target” sequence. The genosensor concept further includes microfabricated devices in which microelectronic components are present in each test site, permitting rapid, addressable detection of hybridization across the array.
The present invention provides a novel flow-through genosensor, in which nucleic acid recognition elements are immobilized within densely packed pores or channels, arranged in patches across a wafer of solid support material. Known microfabrication techniques are available for producing microchannel or nanochannel glass and porous silicon useful as support wafers. Flow-through genosensors utilize a variety of conventional detection methods, including microfabricated optical and electronic detection components, film, charge-coupled-device arrays, camera systems and phosphor storage technology.
The following advantages for the novel flow-through apparatus herein as compared to known flat surface designs are obtained:
(1) improved detection sensitivity due to the vastly increased surface area which increases the quantity of nucleic acid bound per cross sectional area;
(2) minimization of a rate-limiting diffusion step preceding the hybridization reaction (reducing the time required for the average target molecule to encounter a surface-tethered probe from hours to milliseconds), speeding hybridization and enabling mismatch discrimination at both forward and reverse reactions;
(3) enablement of the analysis of dilute nucleic acid solutions because of the ability to gradually flow the solution through the porous wafer;
(4) facilitation of subsequent rounds of hybridization involving delivery of probes to specific sites within the hybridization array;
(5) facilitation of the recovery of bound nucleic acids from specific hybridization sites within the array, enabling the further analysis of such recovered molecules; and
(6) facilitation of the chemical bonding of probe molecules to the surface within each isolated region due to the avoidance of the rapid drying of small droploets of probe solution on flat surfaces exposed to the atmosphere.
Accordingly, the present invention provides an improved apparatus and method                for the simultaneous conduct of a multiplicity binding reactions on a substrate,        which substrate is a microfabricated device comprising a set of discrete and isolated regions on the substrate,        such that each such discrete and isolated region corresponds to the location of one such binding reaction,        in which each such discrete and isolated region contains an essentially homogeneous sample of a biomolecule of discrete chemical structure fixed to such bounded region,        such that upon contact between the substrate and a sample (hereinafter, “test sample”) containing one or more molecular species capable of controllably binding with one or more of the pre-determined biomolecules,        the detection of the bounded regions in which such binding has taken place yields a pattern of binding capable of characterizing or otherwise identifying the molecular species in the test sample.        
The present invention specifically provides novel high-density and ultra-high density microfabricated, porous devices for the conduction and detection of binding reactions. In particular, the present invention provides improved “genosenors” and methods for the use thereof in the identification or characterization of nucleic acid sequences through nucleic acid probe hybridization with samples containing an uncharacterized polynucleic acid, e.g., a cDNA, mRNA, recombinant DNA, polymerase chain reaction (PRC) fragments or the like, as well as other biomolecules.
During the past decade microfabrication technology has revolutionized the electronics industry and has enabled miniaturization and automation of manufacturing processes in numerous industries. The impact of microfabrication technology in biomedical research can be seen in the growing presence of microprocessor-controlled analytical instrumentation and robotics in the laboratory, which is particularly evident in laboratories engaged in high throughput genome mapping and sequencing. The Human Genome Project is a prime example of a task that whose economics would greatly benefit from microfabricated high-density and ultra-high density hybridization devices that can be broadly applied in genome mapping and sequencing.
Hybridization of membrane-immobilized DNAs with labeled DNA probes is a widely used analytical procedure in genome mapping. Robotic devices currently enable gridding of 10,000–15,000 different target DNAs onto a 12 cm×8 cm membrane. Drmanac, R., Drmanac, S., Jarvis, J. and Labat, 1. 1993. in Venter, J. C. (Ed.), Automated DNA Sequencing and Analysis Techniques, Academic Press, in press, and Meier-Ewert, S., Maier, E., Ahmadi, A., Curtis, J. and Lehrach, H. 1993. Science 361:375–376. Hybridization of DNA probes to such filters has numerous applications in genome mapping, including generation of linearly ordered libraries, mapping of cloned genomic segments to specific chromosomes or megaYACs, cross connection of cloned sequences in cDNA and genomic libraries, etc. Recent initiatives in “sequencing by hybridization” (SBH) aim toward miniaturized, high density hybridization arrays. A serious limitation to miniaturization of hybridization arrays in membranes or on flat surfaces is the quantity of DNA present per unit cross sectional area, which (on a two-dimensional surface) is a function of the surface area. This parameter governs the yield of hybridized DNA and thus the detection sensitivity.
Genosensors, or miniaturized “DNA chips” are currently being developed in several laboratories for hybridization analysis of DNA samples. DNA chips typically employ arrays of DNA probes tethered to flat surfaces, e.g., Fodor, S. P. A., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T. and Solas, D. 1991. Science 251:767–773, Southern, E. M., Maskos, U. and Elder, J. K. 1992. Genomics 13:1008–1017, Eggers, M. D., Hogan, M. E., Reigh, R. K., Lamture, J. B., Beattie, K. L., Hollis, M. A., Ehrlich, D. J., Kosicki, B. B., Shumaker, J. M., Varma, R. S., Burke, B. E., Murphy, A. and Rathman, D. D. 1993. Advances in DNA Sequencing Technology, SPIE Conference, Los Angeles, Calif., and Beattie, K., Eggers, M., Shumaker, J., Hogan, M., Varma, R., Lamture, J., Hollis, M., Ehrlich, D. and Rathman, D. 1993. Clin. Chem. 39:719–722, to acquire a hybridization pattern that reflects the nucleotide sequence of the target DNA. The detection limit for hybridization on flat-surface genosensors, as in membrane hybridization, is limited by the quantity of DNA that can be bound to a two dimensional area
Another limitation of these prior art approaches is the fact that a flat surface design introduces a rate-limiting step in the hybridization reaction, i.e., diffusion of target molecules over relatively long distances before encountering the complementary probes on the surface. In contrast, the microfabricated apparatus according to the present invention is designed to overcome the inherent limitations in current solid phase hybridization materials, eliminating the diffusion-limited step in flat surface hybridizations and increasing the cross sectional density of DNA.
Typically microfabricated genosensor devices are characterized by a compact physical size and the density of components located therein. Known microfabricated binding devices are typically rectangular wafer-type apparatuses with a surface area of approximate one cm2, e.g., 1 cm×1 cm. The bounded regions on such devices are typically present in a density of 102–104 regions/cm2, although the desirability of constructing apparatuses with much higher densities has been regarded as an important objective. See Eggers and Beattie, cited above, for discussion of strategies for the construction of devices with higher densities for the bounded regions.
The microfabricated apparatuses as described herein are known to be useful for a variety of analytical tasks, including nucleic acid sequence analysis by hybridization (SBH), analysis of patterns of gene expression by hybridization of cellular mRNA to an array of gene-specific probes, immunochemical analysis of protein mixtures, epitope mapping, assay of receptor-ligand interactions, and profiling of cellular populations involving binding of cell surface molecules to specific ligands or receptors immobilized within individual binding sites. Although nucleic acid analysis is one principal use for such an microapparatus, it is advantageously applied to a broad range of molecular binding reactions involving small molecules, macromolecules, particles, and cellular systems. See, for example, the uses described in PCT Published Application WO 89/10977.
Ordinarily the microfabricated apparatus is used in conjunction with a known detection technology particularly adapted to discriminating between bounded regions in which binding has taken place and those in which no binding has occurred and for quantitating the relative extent of binding in different bounded regions. In DNA and RNA sequence detection, autoradiography and optical detection are advantageously used. Autoradiography is performed using 32P or 35S labelled samples. For traditional DNA sequence analysis applications, nucleic acid fragments are end-labeled with 32P and these end-labeled fragments are separated by size and then placed adjacent to x-ray film as needed to expose the film, a function of the amount of radioactivity adjacent to a region of film. Alternatively, phosphorimager detection methods may be used.
Optical detection of fluorescent-labelled receptors is also employed in detection. In traditional sequencing, a DNA base-specific fluorescent dye is attached covalently to the oligonucleotide primers or to the chain-terminating dideoxynucleotides used in conjunction with DNA polymerase. The appropriate absorption wavelength for each dye is chosen and used to excite the dye. If the absorption spectra of the dyes are close to each other, a specific wavelength can be chosen to excite the entire set of dyes. One particularly useful optical detection technique involves the use of ethidium bromide, which stains duplex nucleic acids. The fluorescence of these dyes exhibits an approximate twenty-fold increase when it is bound to duplexed DNA or RNA, when compared to the fluorescence exhibited by unbound dye or dye bound to single-stranded DNA. This dye is advantageously used to detect the presence of hybridized polynucleic acids.
A highly preferred method of detection is a charge-coupled-device array or CCD array. With the CCD array, a individual pixel or group of pixels within the CCD array is placed adjacent to each confined region of the substrate where detection is to be undertaken. Light attenuation, caused by the greater absorption of an illuminating light in test sites with hybridized molecules, is used to determine the sites where hybridization has taken place. Lens-based CCD cameras can also be used.
Alternatively, a detection apparatus can be constructed such that sensing of changes in AC conductance or the dissipation of a capacitor placed contiguous to each confined region can be measured. Similarly, by forming a transmission line between two electrodes contiguous to each confined region hybridized molecules can be measured by the radio-frequence (RF) loss. The preferred methods for use herein are described in, Optical and Electrical Methods and Apparatus for Molecule Detection, PCT Published Application WO 93/22678, published Nov. 11, 1993, and expressly incorporated herein by reference.
Methods for attaching samples of substantially homogeneous biomolecules of a pre-determined structure to the confined regions of the microapparatus are likewise known. One preferred method of doing so is to attach these biomolecules covalently to surfaces such as glass or gold films. For example, methods for attachments of oligonucleotide probes to glass surfaces are known. A primary amine is introduced at one terminus during the chemical synthesis thereof. Optionally, one or more triethylene glycol units may be introduced therebetween as spacer units. After derivatizing the glass surface in the confined region with epoxysilane, the primary amine terminus of the oligonucleotide can be covalently attached thereto.
See Beattie, et al., cited above, for a further description of this technology for fixing the pre-determined biomolecules in the bounded regions of the microfabricated apparatus.